Fucosylation-deficient cells

ABSTRACT

An isolated nucleic acid encoding an FX protein having a serine at position 79, a lysine at position 90, a leucine at position 136, an arginine at position 211, a serine at position 289, and a combination thereof is provided. Cells having a gene encoding a modified FX protein are provided, wherein the cells exhibit a reduced ability to fucosylate a glycoprotein at a first temperature, but exhibit the ability to fucosylate the glycoprotein at a second temperature. Methods and compositions for making glycoproteins with reduced fucosylation are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.15/987,286 filed May 23, 2018, which is a continuation application ofU.S. Ser. No. 15/378,670 filed Dec. 14, 2016, now U.S. Pat. No.10,006,070, which is a continuation application of U.S. Ser. No.14/963,178 filed Dec. 8, 2015, now U.S. Pat. No. 9,550,823, which is adivisional application of U.S. Ser. No. 13/779,952 filed Feb. 28, 2013now U.S. Pat. No. 9,206,455, which is a divisional application of U.S.Ser. No. 12/791,637 filed Jun. 1, 2010 now U.S. Pat. No. 8,409,838,which claims the benefit under 35 USC Section 119(e) of U.S. ProvisionalApplication Ser. No. 61/183,400 filed Jun. 2, 2009, and U.S. ProvisionalApplication Ser. No. 61/348,858, filed May 27, 2010, each of whichapplications is hereby incorporated by reference.

REFERENCE TO A SEQUENCE LISTING AS A TEXT FILE VIA EFS WEB

This application includes a sequence listing submitted herewith as atext file named 471511_SEQLST.txt created on Dec. 8, 2015, andcontaining 17,981 bytes. The material contained in this text file isincorporated by reference in its entirety for all purposes.

FIELD

The invention relates to a modified mammalian enzyme in the fucosylationpathway, wherein cells bearing the modified mammalian enzyme exhibit areduced ability to fucosylate a protein, and to cells comprising agenetic modification that results in a reduced ability to fucosylate aprotein. The invention includes mammalian cell lines (e.g., CHO lines)that express proteins, including antibodies, with reduced fucosylationas compared to wild-type cell lines. The invention also relates toconditional control of protein fucosylation.

BACKGROUND

Cell lines that are unable to fucosylate proteins are known in the art.A number of loss-of-function mutants that are unable to fucosylateproteins are known, perhaps most notably certain Chinese hamster ovary(CHO) cell mutants selected for resistance to certain lectins. Such celllines are isolated by repeated selection for the inability to bind aparticular lectin, e.g., the Lens culinaris lectin, in the presence of amutagen. Other cell lines reportedly incapable of fucosylating proteins,e.g., antibodies, are known, see, e.g., U.S. Pat. Nos. 7,425,466 and7,214,775 (α1,6-fucosyltransferase, i.e., FUT8 mutant). There remains aneed in the art for cell lines with reduced ability to fucosylateproteins, in particular for cells with reduced fucosylation ability inthe absence of a knockout, and for cells that conditionally fucosylateproteins.

SUMMARY

In one aspect, an isolated modifiedGDP-4-keto-6-deoxy-mannose-3,5-epimerase-4-reductase (FX) protein isprovided, comprising a modification selected from the group consistingof 79S, 90K, 136L, 211R, 289S, and a combination thereof. In oneembodiment, the FX protein comprises a 289S modification. In oneembodiment, the FX protein comprises a 289S modification and at leastone modification selected from the group consisting of 79S, 90K, 136L,211R, and a combination thereof.

In one aspect, a nucleic acid that codes for a modified FX proteinsequence is provided. In a specific embodiment, the nucleic acid is acDNA. In one embodiment, an expression vector or a targeting vectorcomprising the nucleic acid is provided. In one embodiment, the nucleicacid of the targeting vector comprises an intron. In one embodiment, thenucleic acid of the targeting vector comprises a cDNA encoding themodified FX protein. In a specific embodiment, the targeting vectorcomprises a targeting sequence that targets the vector to a locus in ahuman, non-human primate, hamster, mouse, or rat genome.

In one aspect, a cell is provided that comprises a modification to anucleic acid that codes for an FX protein, or that expresses an FXprotein with a modification, wherein the cell does not express or doesnot substantially express a wild-type FX protein. In a specificembodiment, the cell exhibits no more than 10%, no more than 5%, no morethan 2%, or no more than 1% wild-type FX protein as compared with a cellthat lacks the modification.

In one embodiment, the cell comprising the modified FX protein ornucleic acid expresses an Fc-containing glycoprotein, wherein the cellfucosylates no more than 90%, no more than 95%, no more than 96%, nomore than 97%, no more than 98%, or no more than 99% of the glycoproteinas compared with a cell that lacks the modification.

In one aspect, a cell is provided that comprises a modification to anucleic acid that encodes an FX protein, or that expresses an FX proteinwith a modification, wherein the cell lacks or substantially lacks theability to fucosylate a glycoprotein at a first temperature, but doesnot lack or does not substantially lack the ability to fucosylate theglycoprotein at a second temperature.

In one embodiment, the first temperature is about 37° C. In oneembodiment, the second temperature is about 34° C.

In one embodiment, the ability to fucosylate the glycoprotein at thefirst temperature is about 1% to about 10% of the ability to fucosylatethe glycoprotein exhibited by a cell that lacks the modification. In oneembodiment, the ability to fucosylate the glycoprotein at the secondtemperature is about 70%, 80%, 90%, or more as compared with the abilityto fucosylate the glycoprotein by a cell that lacks the modification.

In a specific embodiment, the FX protein modification comprises an aminoacid substitution selected from the group consisting of the followingamino acid substitutions: 90K, 289S, 211R, 136L, 79S, and a combinationthereof. In a specific embodiment, the substitution is 289S.

In one embodiment, the FX protein is from a nonhuman primate (e.g.,Macaca mulatta), a human, a mouse (e.g., Mus musculus), a rat (e.g.,Rattus norvegicus), or a hamster (e.g., Chinese hamster, or Cricetulusgriseus). In a specific embodiment, the FX protein comprises the aminoacid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, orSEQ ID NO:6, and bears one or more modifications (e.g., amino acidsubstitutions) as described herein.

In one embodiment, the nucleic acid codes for a FX protein that is atleast 90% or at least 95% identical to the sequence of SEQ ID Nal, andfurther comprises one or more of the following amino acids at one ormore of the following positions: 79S, 90K, 136L, 211R, and 289S.

In one embodiment, the nucleic acid codes for a FX that is at least 95%identical to the FX of SEQ ID NO:2. In a specific embodiment, the FX hasthe amino acid sequence of SEQ ID NO:2.

In one aspect, a cell is provided, wherein the cell comprises amodification that results in a reduced ability of the cell to fucosylatea glycoprotein, and the modification comprises a mutation or alterationin the sequence of a FX gene that results in the reduced ability tofucosylate the glycoprotein.

In one embodiment, the cell expresses a wild-type fucosylation pathwayenzyme selected from the group consisting of GDP-mannose 4,6-dehydratase(GMD), a wild-type GDP-β-L-fucose pyrophosphorylase (GFPP), a wild-typeα-1,6-fucolysltransferase (FUT8), and a combination thereof.

In one aspect, a mammalian cell capable of fucosylating a protein isprovided, wherein the cell comprises a modification in a FX gene,wherein the modification results in at least a 90% reduction in thecell's ability to fucosylate a protein in comparison to a cell thatlacks the mutation or alteration.

In one embodiment, the reduction is about 90%, 91%, 92%, 93%, 94%, 95%,97%, 98%, or 99% in comparison with a mammalian cell that does notcontain the modification.

In one embodiment, comparison of a modified cell according to theinvention and a cell that does not comprise the modification isconducted under the same or under essentially the same conditions (e.g.,media, temperature, cell density, etc.).

In one embodiment, the cell is selected from a COS, CHO, 293, BHK, HeLa,Vero, a mammalian transfected with adenovirus genes, e.g., AD5 El,including but not limited to an immortalized human retinal celltransfected with an adenovirus gene, e.g., a PER.C6™ cell, and an NS©cell. In one embodiment, the cell is a Chinese hamster ovary (CHO) cell.In a specific embodiment, the CHO cell is a CHO K1 cell.

In one embodiment, the modification is selected from the groupconsisting of the following amino acids: 79S, 90K, 136L, 2118, 289S, anda combination thereof. In a specific embodiment, the substitutioncomprises 289S. In another specific embodiment, the substitutioncomprises 289S and one or more of 79S, 90K, 136L, and 211R.

In one embodiment, the cell comprises an FX gene that encodes a proteincomprising the sequence of SEQ ID NO:1, with one or more amino acidsubstitutions selected from the group consisting of N79S, N90K, P136L,G211R, L289S, and a combination thereof. In a specific embodiment, theamino acid substitution comprises L289S and one or more of N79S, N90K,P136L, and G211R.

In one embodiment, the cell further comprises at least one nucleic acidencoding an immunoglobulin protein. In a specific embodiment, theimmunoglobulin protein is a human protein or a mouse protein. In aspecific embodiment, the immunoglobulin protein comprises animmunoglobulin light chain. In a specific embodiment, the immunoglobulinprotein comprises an immunoglobulin heavy chain. In one embodiment, theimmunoglobulin heavy chain is of an IgG1, IgG2, IgG3, or IgG4 isotype.In one embodiment, the immunoglobulin heavy chain is an IgG1 isotype,e.g., a human IgG1 isotype. In one embodiment, the variable region ofthe heavy and/or light chain comprises a human CDR, in anotherembodiment a mouse CDR, in another embodiment a humanized CDR of a mouseor a non-human primate.

In one embodiment, the cell comprises a nucleic acid encoding a CH2 anda CH3 domain of an immunoglobulin heavy chain. In one embodiment, theimmunoglobulin heavy chain is of an isotype IgG1, IgG2, IgG3, or IgG4.

In one embodiment, the protein is an antigen-binding protein. In aspecific embodiment, the antigen-binding protein is an antibody. Inspecific embodiments, the antibody comprises a heavy chain of an IgA,IgD; IgE, IgG, or IgM isotype. In one embodiment, the antigen-bindingprotein is an antibody of IgG1 isotype.

In one embodiment, the protein is an antibody and only about 5%, 4%, 3%,2%, 1%, 0.5% of the antibody protein made by the cell is fucosylated. Inone embodiment, the amount of antibody protein made that is fucosylatedis measured by overnight deglycosylation of antibody protein with PNGaseF followed by oligosaccharide analysis via HPLC whereinfucosyl-containing oligosaccharides are quantified by integration ofglycan peak area, and, e.g., protein fucosylation is calculated based onglycan peak area. In a specific embodiment, fucosylated glycans areidentified by mass spectroscopy.

In one aspect, a method for making an antigen-binding protein isprovided, the method comprising: (a) providing a cell capable offucosylating a protein, wherein the cell comprises a modification in aFX gene that results in at least a 90% reduction in the cell'scapability to fucosylate a protein; (b) introducing into the cell anucleic acid sequence encoding an antigen-binding protein; (c)maintaining the cell under conditions sufficient to express the nucleicacid sequence to produce the antigen-binding protein; and, (d)recovering the antigen-binding protein expressed by the cell.

In one embodiment, the antigen-binding protein is an antibody. In aspecific embodiment, the antibody is selected from a human antibody, amouse antibody, a chimeric human/mouse antibody, and a non-human primateantibody.

In one embodiment, the cell is a Chinese hamster ovary (CHO) cell.

In one embodiment, the reduction in the cell's capacity to fucosylate aprotein is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% ascompared to a cell that lacks the modification in the FX gene.

In one embodiment, the modification is selected from the groupconsisting of the following amino acids at the following positions: 79S,90K, 136L, 211R, and 289S. In one embodiment, the modification comprises289S and at least one of 79S, 90K, 136L, and 211R.

In one embodiment, the fucosyltransferase gene encodes a proteincomprising the sequence of SEQ ID NO:1, with amino acid substitutionsselected from the group consisting of N79S, N90K, P136L, G211R, andL289S. In one embodiment the modification comprises L289S and at leastone of N79S, N90K, P136L, and G211R.

In one embodiment, the antibody or fragment thereof is a human antibodyor fragment thereof. In a specific embodiment, the antibody is an IgG1isotype, e.g., a human IgG1.

In one embodiment, the recovered antibody has no more that about 5°/hfucosylation as compared to the same antibody made in a wild-type cellthat lacks the modification, in another embodiment, no more than 4%, 3%,2%, 1%, or 0.5% fucosylation as compared to the same antibody made in awild-type cell that lacks the modification.

In one aspect, a cell is provided that expresses a wild-typefucosylation pathway enzyme selected from the group consisting ofGDP-mannose 4,6-dehydratase (GMD), a wild-type GDP-β-L-fucosepyrophosphorylase (GFPP), a wild-type α-1,6-fucolysltransferase (FUT8),and a combination thereof; wherein the cell comprises a modified FXgene, wherein the cell has a reduced ability to fucosylate aglycoprotein as compared to a cell that lacks the modification to the FXgene.

In a specific embodiment, the glycoprotein comprises an Fc. In oneembodiment, the protein is an antibody. In one embodiment, the proteincomprises a sequence of an IgG. In a specific embodiment, the sequenceof an IgG is an IgG1, an IgG2, an IgG3, an IgG4 sequence, or acombination thereof. In a specific embodiment, the protein is anantibody and the antibody comprises an Fc having an IgG1, IgG2, IgG3,and/or IgG4 sequence.

In one embodiment, the cell is selected from CHO, COS, human retinal(e.g., PER.C6™) Vero, or HeLa cell.

In one aspect, a method is provided for making a glycoprotein,comprising expressing a glycoprotein in a mammalian cell, wherein themammalian cell comprises a modified FX gene.

In one embodiment, a method for making a glycoprotein is provided,comprising culturing a glycoprotein-expressing CHO cell in culturemedium under conditions sufficient or the CHO cell to express theglycoprotein, and recovering from the CHO cell or the culture medium theexpressed glycoprotein. In one embodiment, the expressed glycoprotein isno more than about 5% fucosylated. In one embodiment, no more than about4%, 3%, 2%, 1%, or 0.5% fucosylated. In a specific embodiment, thepercent fucosylation is a mole percent of fucose to glycan. In aspecific embodiment, the percent fucosylation is a mole percent offucose to glycoprotein. In a specific embodiment, the molar ratio ofnonfucosylated to fucosylated protein is about 0.90 to 0.10, about 0.91to 0.09, about 0.92 to 0.08, about 0.93 to 0.07, about 0.94 to 0.06,about 0.95 to 0.05, about 0.96 to 0.04, about 0.97 to 0.03, about 0.98to 0.02, or about 0.99 to 0.01.

In one embodiment, the glycoprotein comprises an immunoglobulin CH2 andCH3 region having at position 297 (EU numbering) the following glycanmoiety: GlcNAc(1) bound to the glycoprotein through the N-linkage;GlcNAc(1)-GlcNAc(2)-Mannose(1), wherein Mannose(1) bears a first and asecond moiety, wherein the first moiety consists essentially ofMannose(2)-ManGlcNAc(3); and wherein the second moiety consistsessentially of Mannose(3)-GlcNAc(4). In one embodiment, the carbohydratemoiety further consists essentially of a Gal(1) bound to GlcNAc(4). Inanother embodiment, the carbohydrate moiety further consists essentiallyof a Gal(1) bound to GlcNAc(4) and a Gal(2) bound to GlcNAc(3).

In one embodiment, fucosylated glycoprotein comprises a glycan moietyidentical to the nonfucosylated glycan moiety described in the paragraphimmediately preceding this paragraph, but also bears a fucose moiety atGlcNAc(1).

In one aspect, a genetically modified cell is provided, wherein themodification is to a FX gene, and wherein the modification results inthe cell producing a FX mRNA that encodes an FX protein having at leastone of the following amino acids: 79S, 90K, 136L, 211R, 289S; andwherein the cell exhibits a reduced ability to fucosylate a glycoproteinas compared with a cell that lacks the FX gene modification. In oneembodiment, the mRNA encodes an FX protein comprising a serine atposition 289. In another embodiment, the mRNA encodes an FX protein thatfurther comprises at least one of a 79S, 90K, 136L, 211R.

In one aspect, a genetically modified cell is provided, wherein themodification is to a FX gene, wherein the modification alters a codon ofthe FX gene such that the modified FX gene codes for an FX proteinhaving at least one of the following: a serine at position 79, a lysineat position 90, a leucine at position 136, an arginine at position 211,and a serine at position 289. In one embodiment the FX protein comprisesa serine at position 289 and at least one of a lysine at position 90, aleucine at position 136, and/or an arginine at position 211.

In one embodiment, the cell further expresses an Fc-containing protein.In one embodiment, the Fc-containing protein is an antibody.

In one embodiment, the cell glycosylates the Fc-containing protein, butdoes not substantially fucosylate the glycosylated Fc-containingprotein. In a specific embodiment, the fucosylation is about no morethan 5%, 4%, 3%, 2%, 1%, or 0.5% of the fucosylation of the glycosylatedFc-containing protein as compared to a cell that lacks the FX genemodification.

In one embodiment, the glycosylation comprises a biantennary trimannosylgroup. In one embodiment, the molar ratio of fucose to biantennarytrimannosyl group is no more than about 1:20, 1:25, 1:33, 1:50, 1:100,or 1:200. In one embodiment, the molar ratio of fucose to biantennarytrimannosyl group in the fucosylated Fc-containing protein is no morethan about 1:20, 1:25, 1:33, 1:50, 1:100, or 1:200.

In one embodiment, the Fc-containing protein is an antibody, and theglycosylation comprises a glycan moiety at position 297 of the Fc. Inone embodiment, the molar ratio of fucose to glycan moiety is no morethan about 1:20, 1:20, 1:25, 1:33, 1:50, 1:100, or 200. In oneembodiment, the glycan moiety comprises two tandem GlcNAc residuesfollowed by a biantennary trimannosyl moiety, wherein each of twoterminal mannosyl moieties of the trimannosyl moiety bear one GlcNAcresidue. In one embodiment, the molar ratio of fucose to GlcNAc in theglycan is no more than 1:80, 1:100, 1:133, 1:150, 1:200, 1:400, or1:800.

In one aspect, a modified mammalian cell that ectopically expresses aglycoprotein is provided, wherein the modification comprises a modifiedFX nucleic acid sequence, and the cell comprises a fucose salvagepathway and a de novo fucose synthesis pathway and expresses afunctional FUT 8 protein and a functional GMD protein, wherein the denovo fucose synthesis pathway is incapable of substantially fucosylatinga glycoprotein due to the modification of the FX nucleic acid sequenceat about 37° C., but is capable of substantially fucosylating theglycoprotein at about 34° C.

The individual aspects and embodiments described herein are intended tobe employed alone or in combination with any other aspect or embodiment,unless expressly stated otherwise or unless such combination isdisallowed by the context.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a MacVector™ alignment for FX protein sequence (from top tobottom) of monkey (Macacca mulatta), SEQ ID NO:3; human, SEQ ID NO:4;mouse (Mus musculus), SEQ ID NO:5; rat (Rattus norvegicus), SEQ ID NO:6;CHO (Cricetulus griseus), SEQ ID NO:1; and CHO with an L289S and N90Kmodification (designated cell line 8088), SEQ ID NO:2.

FIG. 2 shows flow cytometry histograms of 3033, 6066, 7077, and 8088cells before and after staining with LCA.

FIG. 3 shows flow cytometry histograms of unstained 4044-1 cells, andhistograms of 4044-1, 6069, 2020, and 2121 cells stained with LCA.

FIG. 4 shows flow cytometry histograms of unstained 5055 cells, andhistograms of 5055, 8088, and 1010 cells stained with LCA.

FIG. 5 shows flow cytometry histograms of 4044-1 and 6066-1 cellscultured at 37° C. and 34° C. before and after staining with LCA,

FIG. 6 shows flow cytometry histograms of 3033, 7077, 8088, and 1010cells cultured in media with and without 5 mM L-fucose, and histogramsof 5055 cells cultured in medium without L-fucose. All cells werestained with LCA.

FIG. 7 shows flow cytometry histograms of 8088 cells stably transfectedwith pR4009, pR4010, and pR4011, and 5055 cells.

FIG. 8 shows glycan separation by HPLC for Ab 3.1 in 8088 cells grown at37° C. in the absence of an external fucose source (1.47% fucosylation).

FIG. 9 shows mass spectrometry results for the glycans of FIG. 8;structures of glycans are presented to the right of each peak. GlcNAcresidues are represented by squares; mannose residues are represented bycircles; galactose residues are represented by diamonds.

FIG. 10 shows glycan separation by HPLC for Ab 3.1 in 8088 cells grownat 37° C. in the presence of 10 mM fucose (95.22% fucosylation).

FIG. 11 shows mass spectrometry results for the glycans of FIG. 10;structures of glycans are presented to the right of each peak. GlcNAcresidues are represented by squares; mannose residues are represented bycircles; galactose residues are represented by diamonds; fucose residuesare represented by triangles.

FIG. 12 shows glycan separation by HPLC for Ab 3.2 in 8088 cells grownat 37° C. in the absence of an external fucose source (5.73%fucosylation).

FIG. 13 shows mass spectrometry results for the glycans of FIG. 12;structures of glycans are presented to the right of each peak. GlcNAcresidues are represented by squares; mannose residues are represented bycircles; galactose residues are represented by diamonds.

FIG. 14 shows glycan separation by HPLC for Ab 3.2 in 8088 cells grownat 7° C. in the presence of 10 mM fucose (95.63% fucosylation).

FIG. 15 shows mass spectrometry results for the glycans of FIG. 14;structures of glycans are presented to the right of each peak. GlcNAcresidues are represented by squares; mannose residues are represented bycircles; galactose residues are represented by diamonds; fucose residuesare represented by triangles.

FIG. 16 summarizes results of mass spectrometry studies on wild-type andlow fucosylation cell lines. GlcNAc residues are represented by squares;mannose residues are represented by circles; galactose residues arerepresented by diamonds; fucose residues are represented by triangles.

DESCRIPTION

The invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. Theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since theinvention is encompassed by the granted claims.

Unless defined otherwise, all terms and phrases used include themeanings that the terms and phrases have attained in the art, unless thecontrary is clearly indicated or clearly apparent from the context inwhich the term or phrase is used. Although any methods and materialssimilar or equivalent to those described can be used in the practice ortesting of the present invention, particular methods and materials arenow described. All publications mentioned are hereby incorporated byreference.

Reference to the singular (e.g., “a” or “the”) is intended to encompassreference to the plural, unless the context clearly indicates thatreference to the plural is excluded.

The term “antibody” includes immunoglobulin molecules comprising fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain comprises a heavychain variable (VH) region and a heavy chain constant region (CH). Theheavy chain constant region comprises three domains, CH1, CH2 and CH3.Each light chain comprises a light chain variable (VL) region and alight chain constant region (CL). The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FRs). Each VH and VL comprisesthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 andHCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3.The term “high affinity” antibody refers to an antibody that has a KDwith respect to its target epitope about of 10⁻⁹ M or lower (e.g., about1×10⁻⁹ M, 1×10 10 M, 1×10⁻¹¹ M, or about 1×10⁻¹² M). In one embodiment,KD is measured by surface plasmon resonance, e.g., BIACORE™; in anotherembodiment, KD is measured by ELISA.

The phrase “binding protein” includes any protein that is capable ofspecifically recognizing a binding partner. Specific recognitiongenerally requires that the binding protein bind its binding partnerwith a dissociation constant (KD) of no higher than a few micromolar,and in most instances desirable binding proteins bind their bindingpartners in the nanomolar range, e.g., in various embodiments on theorder of less than a hundred nanomolar. Most binding proteins describedherein are also Fc-containing proteins, i.e., they comprise a bindingmoiety fused with an Fc that comprises at least a functional portion ofan immunoglobulin CH2 and CH3 region. Typical binding proteins areantibodies, multispecific antibodies (e.g., bispecific antibodies),immunoadhesins, traps (e.g., cytokine traps such as IL-1 traps; VEGFtrap, etc.). Typical binding proteins that are not antibodies bear abinding moiety (e.g., a receptor or fragment thereof, a ligand orfragment thereof, a variation on a canonical immunoglobulin variabledomain, etc.) and an immunoglobulin moiety that frequently comprises aCH2 and a CH3 immunoglobulin domain (or fragment thereof retaining an Fceffector function). Thus, the compositions and methods of the inventioncan be used to make binding proteins (e.g., including immunoadhesins andtraps) that bear an immunoglobulin region that binds an Fc receptorand/or that activates complement (e.g., a functional CH2 and CH3 region)and thereby is capable of mediating ADCC and/or CDC.

Multispecific antibodies may be specific for different epitopes of onetarget polypeptide or may contain antigen-binding domains specific formore than one target polypeptide. Multispecific binding proteins thatare bispecific can be made that comprise two immunoglobulin arms, e.g.,wherein the first arm of an immunoglobulin is specific for a firstepitope, and the second arm of the immunoglobulin is specific for asecond epitope. Other multispecific binding proteins include thosewherein the second arm bears a binding moiety (a ligand or a receptor orbinding fragment thereof) that specifically binds a target that is aprotein or non-protein binding partner.

The phrase “bispecific antibody” includes an antibody capable ofselectively binding two or more epitopes. Bispecific antibodiesgenerally comprise two nonidentical heavy chains, with each heavy chainspecifically binding a different epitope—either on two differentmolecules (e.g., different epitopes on two different antigens) or on thesame molecule (e.g., different epitopes on the same antigen). If abispecific antibody is capable of selectively binding two differentepitopes (a first epitope and a second epitope), the affinity of thefirst heavy chain for the first epitope will generally be at least oneto two or three or four or more orders of magnitude lower than theaffinity of the first heavy chain for the second epitope, and viceversa. Epitopes specifically bound by the bispecific antibody can be onthe same or a different target (e.g., on the same or a differentprotein). Bispecific antibodies can be made, for example, by combiningheavy chains that recognize different epitopes of the same antigen. Forexample, nucleic acid sequences encoding heavy chain variable sequencesthat recognize different epitopes of the same antigen can be fused tonucleic acid sequences encoding the same or different heavy chainconstant regions, and such sequences can be expressed in a cell thatexpresses an immunoglobulin light chain. A typical bispecific antibodyhas two heavy chains each having three heavy chain CDRs, followed by(N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and aCH3 domain, and an immunoglobulin light chain that either does notconfer epitope-binding specificity but that can associate with eachheavy chain, or that can associate with each heavy chain and that canbind one or more of the epitopes bound by the heavy chainepitope-binding regions, or that can associate with each heavy chain andenable binding or one or both of the heavy chains to one or bothepitopes.

One example of a bispecific binding protein format employs a firstimmunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein thefirst and second Ig CH3 domains differ from one another by at least oneamino acid, and wherein at least one amino acid difference reducesbinding of the bispecific antibody to Protein A as compared to abi-specific antibody lacking the amino acid difference. In oneembodiment, the first Ig CH3 domain binds Protein A and the second IgCH3 domain contains a mutation that reduces or abolishes Protein Abinding such as a 435R modification (by EU numbering; 95R by IMGT exonnumbering). The second CH3 may further comprise a 436F modification (byEU numbering; 96F by IMGT numbering). Further modifications that may befound within the second CH3 include 356E, 358M, 384S, 392N, 397M, and4221 (by EU numbering; 16E, 18M, 44S, 52N, 57M, and 821 by IMGTnumbering). In this format, the first Ig CH3 domain is fused to a firstbinding moiety (e.g., a first Ig variable domain that specifically bindsa first epitope), and the second Ig CH3 domain is fused to a secondbinding moiety (e.g., a second Ig variable domain that specificallybinds a second epitope, wherein the first and the second epitopes aredifferent).

The term “cell” includes any cell that is suitable for expressing arecombinant nucleic acid sequence. Cells include eukaryotes (single-cellor multiple-cell), yeast cells (e.g., S. cerevisiae, S. pombe, P.pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9,SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.),non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. Cells that do not naturally comprise apathway for fucosylation may be genetically modified to contain one(see, e.g., US Patent Application Publication No. 201010028951A1), andthe cell can be modified to employ a FX gene that is modified asdescribed herein.

In some embodiments, the cell is a human, monkey, ape, hamster, rat, ormouse cell. In some embodiments, the cell is eukaryotic and is selectedfrom the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO),COS (e.g., COS-7), syrian hamster, rat myleloma, mouse myeloma (e.g.,SP2/0, NS0), retinal cell, Vero, CV1, kidney (e.g., HEK293, 293 EBNA,MSR 293, MDCK, HaK, BHK, BHK21), HeLa, HepG2, WI38, MRC 5, Colo205, HB8065, HL-60, Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell,C127 cell, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, a humanmyeloma cell, tumor cell, a human lymphoma cell (e.g., a Namalwa cell)and a cell line derived from an aforementioned cell. In someembodiments, the cell comprises one or more viral genes, e.g. the cellis a retinal cell that expresses a viral gene (e.g., a PER.C6™ cell).

The phrase “Fc-containing protein” includes antibodies, bispecificantibodies, immunoadhesins, and other binding proteins that comprise atleast a functional portion of an immunoglobulin CH2 and CH3 region. A“functional portion” refers to a CH2 and CH3 region that can bind an Fcreceptor (e.g., an FcγR or an FcRN), and/or that can participate in theactivation of complement. If the CH2 and CH3 region contains deletions,substitutions, and/or insertions or other modifications that render itunable to bind any Fc receptor and also unable to activate complement,the CH2 and CH3 region is not functional.

Fc-containing proteins can comprise modifications in immunoglobulindomains, including where the modifications affect one or more effectorfunction of the binding protein (e.g., modifications that affect FcγRbinding, FcRN binding and thus half-life, and/or CDC activity). Suchmodifications include, but are not limited to, the followingmodifications and combinations thereof, with reference to EU numberingof an immunoglobulin constant region: 238, 239, 248, 249, 250, 252, 254,255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285,286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307,308, 309, 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330,331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359,360, 361, 362, 373, 375, 376, 378, 380, 382, 383, 384, 386, 388, 389,398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and 439. Forexample, and not by way of limitation, the binding protein may exhibitenhanced serum half-life and have a modification at positions 252, 254,and 256; or a modification at 428 and/or 433 and/or 434; or amodification at 250 and/or 428; or a modification at 307 or 308, and434.

The term “FX” refers to a protein that exhibitsGDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase activity or to anucleic acid sequence that codes for a protein havingGDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase activity. Mostexamples described herein refer to a wild-type C. griseus FX or a C.griseus FX that is modified according to the invention. However, “FX” isnot limited to reference to a CHO cell. As shown in FIG. 1, an alignmentof Macaca mulatta, human, Mus musculus, Rattus norvegicus FX sequencesreveal a very high degree of conservation, i.e., FX sequences fromvarious organisms are very, very similar. Based on this high degree ofidentity, it is to be expected that minor sequence differences thatexist between these species will not substantially affect FX activity.Differences between the CHO FX sequence (SEQ ID NO:1) and sequences frommonkey (SEQ ID NO:3), human (SEQ ID NO:4), mouse (SEQ ID NO:5), and rat(SEQ ID NO:6) include the following: 5H, 8M, 21K, 37D, 51T, 55R, 59E,62R, 93M, 106A, 1070, 138N, 161Y, 167S, 177Y, 201S, 202S, 202D, 212N,225Q, 235S, 266H, 266N, 266S, 273T, 274S, 280F, 287S, 291T, 291S, 2970,310D, 314E. For the 321-amino acid wild-type CHO FX (SEQ ID NO:1), anyone of monkey, human, mouse, and rat FX sequences can be recapitulatedby selecting from 31 different substitutions, or 31/321×100=9.6% of thewild-type CHO FX sequence. Thus, a modified FX of the invention includesa wild-type CHO FX (e.g., SEQ ID NO:1) or an FX having at least a 90.4%identity with a wild-type CHO FX, and also bearing a substitutionselected from the group consisting of N79S, N90K, G211R, and L289S. Forless deviations from SEQ ID NO:1, a modified FX of the invention is atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical with SEQID NO:1 and bears at least one modification selected from the groupconsisting of N79S, N90K, P136L, G211R, and L289S, e.g., bearing anL289S modification. A person of skill would expect that one or more of asmall insertion or one or more of a small deletion that includes atleast one of position 79, 90, 136, 211, and 289 would most likely alsoprovide advantages associated with an embodiment of the invention (e.g.,a cell bearing the modified gene would exhibit reduced fucosylation of aglycoprotein).

In a specific embodiment, the FX comprises a first substitution that is289S and one or more of the second substitutions.

The phrase “low fucosylation” or “reduced fucosylation” refers to alowered or reduced ability of a modified cell to fucosylate aglycoprotein as compared with a normal or wild-type cell. Theglycoprotein may be an endogenous glycoprotein. More typically, thenucleic acid modification is made in a cell that is used to express aheterologous glycoprotein, e.g., a cell that expresses a binding protein(e.g., an antibody or bispecific antibody or an immunoadhesin or otherFc-containing glycoprotein) ectopically. For example, a CHO or PERC.6™cell line modified according to the invention, which also expresses ahuman antibody, e.g., a human IgG1 antibody.

In general, reference to “low fucosylation” or “reduced fucosylation”with respect to a glycoprotein does not refer to a single glycoproteinmolecule having less fucose residues attached to it. Rather, referenceis made to a glycoprotein preparation prepared from cells, and theglycoprotein preparation comprises a population of individualglycoprotein molecules, with members of the population having differentglycosylation features. For purposes of illustration and not limitation,for an IgG1 antibody expressed in a modified CHO cell according to theinvention, “low fucosylation” or “reduced fucosylation” refers to asmaller number of individual glycoproteins having a fucose residue on anN-linked GlcNAc residue of a glycan at position 297 of the Fc. Such “lowfucosylation” or “reduced fucosylation” can be characterized in avariety of ways (see elsewhere herein), but reference is in each case toa relatively low (or reduced) number of the glycoproteins of thepopulation having fucose residues on them as compared to a population ofthe same glycoprotein made in a cell line that lacks a modification inaccordance with the invention.

By way of illustration, if a glycoprotein made in accordance with theinvention is 1% fucosylated as compared with the same glycoprotein madewith a wild-type cell, only 1% of the molecules of Fc-containing proteinare fucosylated in the inventive cell as compared with the amount offucosylation observed in a corresponding wild-type cell (arbitrarily setto 100%, whether or not all of the molecules of Fc-containing proteinare fucosylated in the wild-type cell under the same conditions).

In a “low fucosylation” or “reduced fucosylation” cell according to theinvention, fucosylation of a glycoprotein is reduced about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% in comparison with a cell thatdoes not contain the modification. In a specific embodiment, thereduction is about 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, or 99.9% in comparison with a cell that does not contain themodification. In another specific embodiment, the reduction is about98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, or 98.9% incomparison with a cell that does not contain the modification. Inanother specific embodiment, the reduction is about 97.1%, 97.2%, 97.3%,97.4%, 97.5%, 97.6%, 97.7%, 97.8%, or 97.9% in comparison with a cellthat does not contain the modification. In another specific embodiment,the reduction is about 96.1%, 96.2%, 96%, 96.4%, 96.5%, 96.6%, 96.7%,96.8%, or 96.9% in comparison with a cell that does not contain themodification. In another specific embodiment, the reduction is about95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, or 95.9% incomparison with a cell that does not contain the modification. Inanother specific embodiment, the reduction is about 94.1%, 94.2%, 94.3%,94.4%, 94.5%, 94.6%, 94.7%, 94.8%, or 94.9% in comparison with a cellthat does not contain the modification.

A glycoprotein preparation made in a cell according to the invention isfucosylated only about 5%, about 4%, about 3%, about 2%, about 1%, about0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1% of the amount offucosylation of the same glycoprotein made in a cell that does notcontain the modification.

Another way to characterize a glycoprotein preparation from a “lowfucosylation” or “reduced fucosylation” cell is by the ratio offucosylated to non-fucosylated glycoprotein in the glycoproteinpreparation made by the cell. For example, a glycoprotein preparationmade by a modified cell has a ratio of fucosylatedglycoprotein:nonfucosylated glycoprotein that is about 1:10 through1:15, 1:15 through 1:20, 1:20 through 1:40, 1:40 through 1:60, 1:60through 1:80, 1:80 through 1:100, or 1:100 through 1:150.

Another way to characterize a glycoprotein preparation from a “lowfucosylation” or “reduced fucosylation” cell is by the relative weightpercent of nonfucosylated glycoprotein (as compared with total, i.e.,fucosylated and nonfucosylated, glycoprotein). For example, aglycoprotein preparation made by a modified cell has a percent ofnonfucosylated glycoprotein that is about 90%, about 95%, about 96%,about 97%, about 98%, about 99%, or about 99.5% as compared with thesame glycoprotein preparation from a cell that lacks the modification.

Another way to characterize a glycoprotein preparation from a “lowfucosylation” or “reduced fucosylation” cell is by the relative amountof fucose to glycan or relative amount of fucose to glycan component ofthe glycoprotein preparation. For example, in the case of Fc-containingproteins (e.g., antibodies), the glycosylation comprises a glycan atposition 297, and the glycan comprises a biantennary trimannosyl moiety.In one embodiment, the molar ratio of fucose to glycan moiety is no morethan about 1:20, 1:20, 1:25, 1:33, 1:50, 1:100, or 1:200. In oneembodiment, the ratio of fucose to biantennary trimannosyl moiety is nomore than about 1:20, 1:25, 1:33, 1:50, 1:100, or 1:200. In oneembodiment, the molar ratio of fucose to biantennary trimannosyl moietyin the fucosylated Fc-containing protein is no more than about 1:20,1:25, 1:33, 1:50, 1:100, or 1:200. In one embodiment, the glycan moietycomprises two tandem GlcNAc residues followed by a biantennarytrimannosyl moiety, wherein each of two antennary terminal mannosylmoieties of the trimannosyl moiety bear one GlcNAc residue. In oneembodiment, the molar ratio of fucose to GlcNAc in the glycan is no morethan 1:80, 1:100, 1:133, 1:150, 1:200, 1:400, or 1:800.

In one embodiment, the amount of antibody protein made that isfucosylated is measured by overnight deglycosylation of antibody proteinwith PNGase F followed by oligosaccharide analysis via HPLC whereinfucosyl-containing oligosaccharides are quantified by integration ofglycan peak area, and, e.g., protein fucosylation is calculated based onglycan peak area. The identity (and composition) of the glycan can bedetermined (and/or quantified) by any suitable method (e.g., massspectroscopy).

The phrase “wild-type” includes reference to a cell or an activity thatis not modified according to the invention, e.g., a cell that does notcontain a modified FX nucleic acid sequence or modified FX protein.“Wild-type” FX activity includes reference to any parameter of activity(e.g., enzyme activity) that is exhibited by a natural or non-modifiedFX gene or protein. In comparing “wild-type” activity of an FX proteinand activity of a modified FX protein, the “wild-type” FX protein andthe modified FX protein are isolated in substantially the same mannerfrom substantially the same source (e.g., same cell type, same organism)and compared under substantially the same conditions. In comparing“wild-type” FX activity and modified FX activity between wild-type cellsand modified cells, FX activity is preferably measured undersubstantially the same or substantially similar conditions, with anidentical or substantially identical glycoprotein,

Overview

Modified FX nucleic acid and protein sequences are provided, whereinmodification results in a cell that is unable to support proteinfucosylation in the absence of an external fucose source at a level thata cell bearing lacking the modification can support. The cells exhibit asubstantially reduced ability to fucosylate glycoproteins (in theabsence of a fucose source) at one temperature due to a disruption in anenzyme activity of the de novo pathway for synthesis of the substratefor glycoprotein fucosylation, GDP-L-fucose. At another (higher)temperature, the reduction in the cell's ability is minimal orunsubstantial.

The enzyme GDP-4-keto-deoxy-mannose-3,5,-epimerase-4-reductase (FX),participates in the de novo pathway of GDP-L-fucose synthesis, formingGDP-L-fucose from GDP-4-keto-6-deoxymannose. The resulting GDP-L-fucosecan be used by a cell to make fucosylated proteins, includingfucosylated antibodies. Since GDP-L-fucose synthetase participates inthe de novo pathway, reduction in fucosylation in cells that lacksufficient FX activity can be rescued by a salvage pathway. The salvagepathway requires fucose, which is acted upon through the salvage pathwayto form GDP-L-fucose, a substrate for protein fucosylation.

Certain modifications in FX result in the inability of a cell having amodified FX gene to sustain protein fucosylation in the absence of afucose source, such that a cell bearing the modified enzyme andexpressing, e.g., a recombinant antibody, exhibits a substantialreduction in the ability to fucosylate the antibody, as compared to awild-type cell bearing wild-type FX gene.

The inability to sustain a sufficient rate or level of proteinfucosylation due to the FX gene modifications described herein issubstantially temperature-dependent. In particular, cells bearing amodified FX gene exhibit a substantial inability to sustain glycoproteinfucosylation at about 37° C. (e.g., 7% fucosylation of a human IgG1isotype antibody), which is substantially relieved at about 34° C.(e.g., 70% fucosylation of the same antibody; see Table 3). Cells thathave only a wild-type FX gene, in contrast, do not exhibit a largedifference in the ability to sustain glycoprotein fucosylation at 37° C.as compared to 34° C.

De Novo and Salvage Pathways to GDP-Fucos.

GDP-fucose is a central metabolite in the glycoprotein fucosylationpathway; it is the fucose donor in glycoprotein fucosylation. All knownfucosyltransferases of interest that can fucosylate a glycoproteincomprising an Fc use GDP-fucose. Thus, for efficient glycoproteinfucosylation, a sufficient pool of GDP fucose must be generated andmaintained at a sufficient level to match glycoprotein production.

There are two major pathways to GDP-fucose: a de novo synthesis pathwayand a salvage pathway. In many mammalian cells, GDP-fucose can be madefrom an externally supplied carbon source (e.g., glucose) by a de novofucosylation pathway. In the de novo pathway for fucosylation, glucoseenters the cell through a transporter, and is converted toD-mannose-1-phosphate. D-mannose-1-phosphate is then converted byD-mannose-1-phosphate guanylyltransferase into GDP-mannose. GDP-mannoseis converted to GDP-4-keto-6-deoxy-mannose by GDP mannose 4,6-dhydratase(GMD). GDP-4-keto-6-deoxy-mannose is converted to GDP fucose byGDP-4-keto-6-deoxy-mannose-3,5-epimerase-4-reductase (FX). GDP-fucose isa potent feedback inhibitor of GMD. GDP-fucose enters the Golgiapparatus through a GDP-fucose transporter. Once in the Golgi, theGDP-fucose is a substrate for α1,6-fucosyltransferase, which fucosylatesglycoproteins.

In many mammalian cells, a salvage pathway for generating GDP-fucosefrom externally supplied fucose also exists. In the salvage pathway,fucose is transported into the cell and phosphorylated to formfucose-1-phosphate, which can be converted to GDP-fucose. The GDP-fucoseis transported into the Golgi and is available as a substrate for theα1,6-fucosyltransferase. Fucose transport into the cell is presumably byfacilitated diffusion and by lysosomal transport, and the salvagepathway appears to be universal in mammalian cells (see, e.g., Beckerand Lowe (2003) Fucose: biosynthesis and biological function in mammals,Glycobioiogy 13(7):41R-53R).

Thus, in the absence of a fucose source, cells fucosylate glycoproteinsusing fucose generated by the de novo synthesis pathway. In the presenceof fucose, cells fucosylate glycoproteins using fucose transported intothe cell. Therefore, if the de novo synthesis pathway is blocked ordamaged, glycoprotein fucosylation can still occur, but only in thepresence of a fucose source.

The compositions and methods described enable cell lines that provide aconditional block in a pathway for fucosylating a glycoprotein byproviding a genetically modified cell having a modification in an FXnucleic acid sequence. In cells that contain both a de MVO and a salvagepathway, the cells provide enhanced versatility. In such cells in theabsence of an external source of fucose, fucosylation of a glycoproteincan be substantially reduced at one temperature, but not substantiallyreduced at a second temperature. Alternatively, essentially wild-typerates or levels of glycoprotein fucosylation can be switched on byproviding an external source of fucose, without regard to thetemperature at which the cell is maintained.

FX Modifications

An isolated nucleic acid that encodes a modified FX protein sequence isprovided. The isolated nucleic acid encodes an FX protein comprising anamino acid modification selected from the group consisting of 79S, 90K,136L, 211R, 289S, and a combination thereof. In one embodiment, theisolated nucleic acid encodes an FX protein comprising a serine atposition 289, in another embodiment comprising at least one of a 79S,90K, 136L, and/or 211R.

The nucleic acid encoding the modified FX protein is used in anysuitable form. Suitability of the form of the nucleic acid depends uponits use. For example, suitable forms include a cDNA that can be used inan expression vector for extrachromosomal expression in a cell, or whichcan be integrated (at a specific location, or randomly) into a genome ofa cell. Suitable forms also include a genomic sequence, which ismodified to encode the substitution(s) described herein. Suitable formsalso include, for example, a targeting sequence (e.g., one or moretargeting arms) for targeting the nucleic acid to a specific location ina genome, e.g., to replace one or both alleles of a native FX gene.Suitable forms include targeting vectors that target the nucleic acid toa specific location in a cell, e.g., to an FX locus, for replacement ofan endogenous FX nucleic acid sequence with an FX nucleic acid sequenceaccording to the invention. Modification of an endogenous FX sequencecan be made at one or both alleles of the cell.

Cell Lines with Low or Reduced Fucosylation

Compositions and methods for low fucosylating cell lines are provided.The compositions include nucleic acids and proteins that, when presentin a cell that lacks or substantially lacks a native or wild-type FXactivity, confer upon the cell a reduced ability to fucosylate aglycoprotein, e.g., an Fc-containing glycoprotein such as, e.g., anantibody. In various embodiments, such cells include cells that exhibita substantially reduced ability to fucosylate a glycoprotein at a firsttemperature (e.g., about 37° C.), but retain the ability to fucosylatethe glycoprotein at a second temperature (e.g., about 34° C.). Thus,cells are provided that can be grown at a first temperature that isfucosylation-inhibiting, and growth conditions can be changed to asecond temperature that is fucosylation-permissive.

Low fucosylating cell lines can be made using any suitable cells inconjunction with compositions and methods described herein. For example,and not by way of limitation, any cell lines commonly employed in themanufacture of biopharmaceuticals can be used.

Certain methods and compositions for rendering such cell lines useful aslow fucosylation cell lines are described herein; others are obvious orreadily apparent by those skilled in the art in light of thisdescription. Human cells (e.g., HeLa, PERC.6™, etc.), CHO cells, mousecells, etc. can be genetically modified as described herein to generatea useful cell line. In the case of CHO cells, e.g., a useful lowfucosylation cell line can be made by modifying a single allele of theFX gene due to the observation that CHO FX activity appears to befunctionally haploid. In the case of other cells that exhibit afunctional diploidy at the FX locus, on the other hand, can bemanipulated by replacing one FX allele with a modified FX nucleic acidsequence as described herein and knocking out the second (wild-type)allele, or replacing both FX alleles with a modified FX nucleic acidsequence as described herein.

Resulting cells include those whose FX activity is wholly orsubstantially characterized as low fucosylating. That is, the cell neednot be completely devoid of a wild-type FX protein or a wild-type FXgene; however, the cell—under an appropriate condition or set ofconditions (e.g., a selected temperature)—should be unable orsubstantially unable to fucosylate a glycoprotein (e.g., anFc-containing glycoprotein) at anywhere near the level that acorresponding cell with altogether normal FX activity can fucosylate thesame glycoprotein.

Comparison of a cell according to the invention and a cell that does notcontain the modification is conducted under the same or underessentially the same conditions (e.g., media, temperature, cell density,etc.). For example, in various embodiments the cell will exhibit anability to fucosylate no more than 10% to no more than 1% of the abilityto fucosylate that a wild-type cell exhibits. This comparison can bedone, for example, by preparing a cell having the modification(s)described herein, and comparing the level of fucosylation of aglycoprotein expressed by the cell (e.g., expression of an antibody froman expression construct in the cell) to the level of fucosylation of thesame glycoprotein expressed by a wild-type cell. For the comparison, thecells are grown at the same temperature and under the same conditions.Fucosylation level of the glycoprotein can be ascertained using anysuitable analytical method known in the art for quantitating the amountof fucose present in a glycoprotein preparation.

In determining how much of the glycoprotein is fucosylated, the amountof fucose is compared with either the amount of total glycoprotein orwith the amount of glycan obtained from the protein.

Fucosylation-Deficient Cell Lines

A number of mammalian cell lines that are altogether unable tofucosylate glycoproteins have been isolated. Development offucosylation-deficient cell lines has been largely fueled by the need tomake antibodies that lack fucosylation. Antibodies that lackfucosylation can mediate antibody-dependent cell-mediated cytotoxicity(ADCC) far better than fucosylated antibodies, due to altered binding toan Fc receptor. Antibodies that mediate ADCC better are therefore highlydesirable, particularly antibodies that comprise variable regions thattarget tumor cells. Cells that are unable to fucosylate glycoproteinsare thus widely used in the development and manufacture of antibodiesfor therapeutic uses.

Two fucosylation pathway knockouts have been developed that result in acell's inability to fucosylate a glycoprotein. Knockout ofα1,6-fucosyltransferase (FUT8) results in the inability to transferGDP-fucose to a glycoprotein. Knockout of GDP mannose 4,6-dehydratase(GMD) results in the inability to make GDP-4-keto-6-deoxy-mannose fromGDP-mannose in the de novo pathway.

Fucosylation knockouts downstream of GDP-fucose formation, e.g.,α1,6-fucosyltransferase knockouts, cannot resort to the salvage pathwayto fucosylate glycoproteins in the presence of an external fucosesource. This is because the block is distal to the formation ofGDP-fucose, the metabolite at which the de novo and salvage pathwaysmeet. Therefore, feeding cells that have such a knockout with fucosewill not rescue glycoprotein fucosylation. Thus, α1,6-fucosyltransferaseknockouts offer no simple route to selectively manipulating a cell'sability to fucosylate a glycoprotein.

Fucosylation knockouts upstream of GDP-fucose formation, e.g., GMDknockouts, can theoretically resort to the salvage pathway to fucosylateglycoproteins. This is because the block occurs before formation ofGDP-fucose. Feeding such cells with fucose will theoretically rescueglycoprotein fucosylation. Cell lines that contain knockouts, however,lack versatility.

The inventors have found that a selective disruption of the de novofucosylation pathway prior to formation of GDP-fucose will generate acell line with a defect that is rescued by providing a source of fucose,or rescued by maintaining the cells under conditions that are permissivefor fucosylation. The inventors have modified cells to have a defect inthe de novo pathway upstream of GDP-fucose and that can be grown in theabsence of fucose under a first condition and exhibit a substantiallyreduced ability to fucosylate a glycoprotein, whereas under a secondcondition the cells can effectively fucosylate a glycoprotein even inthe absence of an external fucose source. Such a particularly versatilecell line presents the option of turning fucosylation on or off in thecell line by controlling the availability of an external source offucose (or a suitable fucose precursor) and/or growing cells under afucosylation-permissive condition or a fucosylation-deficient condition.

The inventors have selectively disrupted the de novo pathway forGDP-fucose synthesis by generating a mutated FX nucleic acid sequence.The FX protein is a bifunctional epimerase-reductase that epimerizes theC3 hydroxyl and the 05 methyl groups of mannose, formingGDP-4-keto-6-deoxygalactose. An NADPH-dependent reductase activity ofthe bifunctional enzyme then reduces the keto moiety to form GDP-fucose.The FX gene is highly conserved, which is reflected in the high degreesof identity and similarity in FX proteins. See, e.g., Becker and Lowe(2003) Fucose: biosynthesis and biological function in mammals,Glycobiology, 13(7):41R-53R. Thus, the data presented in connection withFX mutations in CHO cells is applicable to corresponding FXmodifications in a wide variety of cells.

The selective FX disruption provides an enhanced versatility, whichallows a practitioner to disfavor, or inhibit, fucosylation bymaintaining a culture at a first temperature; but allow fucosylation bymaintaining the culture at a second temperature. In various embodimentsthis illustrated by a modified FX gene, wherein the modificationcomprises a modification selected from the group consisting of (for aCHO FX protein) L289S, N79S, N90K, P136L, G211R, and a combinationthereof. In various embodiments, the FX modification consistsessentially of a modification selected from the group consisting ofL289S, L289S/N90K, L289S/G211R, L289S/P136L, L289S/N79S, and acombination thereof.

As those skilled in the art know, certain cells that are diploid displayphenotypes that reflect activity of only one of two alleles atparticular loci, e.g., CHO cells, and with respect to those loci appearto be functionally haploid (or hypodiploid) from a phenotypicperspective. In such cells, modification of even a single allele asdescribed herein may result in a phenotype that essentially reflects theactivity of the modified allele, even in cases where the phenotype isnot a dominant phenotype. For example, in CHO cells, modification asdescribed herein of a single allele will likely result in a FX phenotypeessentially as described herein, presumably due to nonexpression (orhypoexpression) of the wild-type FX allele.

In various embodiments, the FX is an FX that is not from a CHO cell, andthe modification comprises a modification selected from the groupconsisting of a modification that corresponds in the non-CHO FX to theCHO modifications listed above. Corresponding modifications can beidentified by aligning the CHO FX protein sequence with any other FXsequence of interest (with or without gaps in the alignment) using,e.g., a general purpose multiple sequence alignment algorithm such asClustalW with default parameters (e.g., for human (Accession No.AAC50786) and G. griseus (Accession No. AAM91926) FX, using MacVector™v. 10.0.2, pairwise: Gannet matrix at slow alignment speed, open gappenalty=10.0, extend gap penalty=0.1; multiple: Gonnet series, open gappenalty=10.0, extend gap penalty=0.2, delay divergent=30%, gapseparation distance=4, no end gap separation, residue-specificpenalties, and hydrophilic penalties (hydrophilic residues GPSNDQEKR)).

As a practical matter, aligning a subject sequence against SEQ ID NO:1or SEQ ID NO:1 and 3-6 in MacVector™ using the pairwise alignmentdefault parameters will identify corresponding positions in the subjectsequence at which to make modifications at positions equivalent to theCHO N79, N90, P136, G211, and L289, positions.

Glycoproteins

The compositions and methods can be used to modify the fucosylatingability of cells to achieve low fucosylation of any glycoprotein ofinterest. Although the majority of this disclosure refers to advantagesof reduced fucosylation in antibodies, the benefits of the invention arenot limited to antibodies. Any binding protein that bears an Fc—andthere are many, many types of such binding proteins—can be made usingthe compositions and methods of the invention.

A typical glycoprotein that can be made with the invention is anantibody (e.g., a human, mouse, or humanized antibody) that isglycosylated and, under normal conditions in a wild-type cell,fucosylated. Examples include, by way of illustration and not by way oflimitation, human antibodies of the IgG1, IgG2, and IgG4 subtypes.Glycoforms of such antibodies include those with a glycan moiety atposition 297. Typical glycoforms at position 297 include an N-linkedGlcNAc, followed by a GlcNAc, followed by a biantennary trimannosylmoiety, followed by (on each of two mannosyl moieties of the biantennarytrimannosyl moiety) one or more GlcNAc residues, optionally followed bya galactose residue on one or more of the GlcNAc residues attached tothe antennae of the biantennary trimannosyl moiety. Fucosylation of theglycan normally occurs at the N-linked initial GlcNAc residue, where(normally) a single fucose residue is linked via a fucosyltransferase tothe 297-glycolated antibody. In various embodiments, the molar ratio orpercent or extent of fucosylation of the antibody is measured withrespect to this fucose residue in relation to the amount (or moles) ofantibody and/or the amount (or moles) of glycan or glycan substituent(e.g., relative moles of fucose:antibody, or to fucose:GlcNAc orfucose:mannose or fucose:trimannosyl moiety or fucose:galactose of the297-linked glycan, in an antibody preparation obtained from wild-typecells or from cells comprising a modified FX nucleic acid sequence inaccordance with the invention).

Low-Fucosylation CHO Lines

A low fucosylation CHO line was constructed from CHO K1 cells adapted togrow in suspension in a serum-free bioreactor medium. The CHO line(designated line 6066) contained an L2893 substitution in the CHO FXgene. A recombinant antibody that is a human IgG1 that specificallybinds an interleukin receptor (Antibody 1) and a recombinant antibodythat is a human IgG1 that specifically binds a cell surface protein ofan immune cell (Antibody 2) were made in the cell line, and in acorresponding CHO cell line that lacks the FX modification (designatedline 4044) as described in the examples. Cells were grown for three daysin shakers or 12 days in a bioreactor (each at 37° C.).

Cells that bore the FX gene modification and expressed Antibody 1fucosylated only about 6.14 or 6.86% (12 days) or about 7 or 8% (threedays) of Antibody 1, whereas in the absence of the FX modification cellsfucosylated about 89.3% (3 days) or about 85.8% (12 days) (Table 1).

Cells that bore the FX gene modification and expressed Antibody 2fucosylated Antibody 2 only about 3.6%, whereas in the absence of the FXgene modification cells fucosylated about 95% (3 days) or about 76.8%(12 days) (Table 1).

Another low fucosylation CHO line was made from CHO K1 cells thatcontained an L289S and a N90K modification of the CHO FX gene(designated line 8088). Antibody 1 expressed in these cells exhibitedonly about 0.96% fucosylation (3 days) or 0.71% fucosylation (12 days)(Table 2).

Another low fucosylation CHO line was made from CHO K1 cells (from6066-1 cells, which have an L289S FX gene modification) that contained aP136L substitution (designated line 2121). These cells expressedAntibody 1 that was only 0.82% fucosylated at 3 days (Table 2).

Two further low fucosylation CHO lines were made from CHO K1 cells (from6066-1 cells, which have an L289S FX gene modification) that contained aN79S substitution (designated lines 2020 and 6069). These cellsexpressed Antibody 1 that was only 0.94% fucosylated at 3 days (2020) oronly 0.86% fucosylated at 3 days (6069) (Table 2).

Temperature dependence of fucosylation for Antibody 1 was tested usingcell lines 4044-1 (no FX gene modification) and cell line 6066-1 (L289SFX gene modification), Cell line 6066-1 exhibited only 7% fucosylationat 37° C., and about 70% fucosylation at 34° C. Cell line 4044-1exhibited about the same fucosylation (95-96%) at both 37° C. and 34°C.,

Two further low fucosylation cell lines were made from cell line 8088(L289S and N90K) that expressed two different antibodies to the samegrowth factor receptor, Ab 3.1 and Ab 3.2. After growing for three daysat 37° C. in the presence (salvage pathway) or absence (de novo pathway)of fucose, glycan composition and fucose content of the glycan wasdetermined. In the absence of fucose, the cell lines produced only about1.87% or 5.73% fucosylation, whereas in the presence of an externalfucose source fucosylation was restored to at least 95.22% or 95.63%.

EXAMPLES Example 1: CHO Cell Lines

A variety of CHO cell lines, isolated directly or indirectly from CHO K1cells, are described herein.

RGC10 Cells. The CHO cell line 3033 was generated from CHO K1 cells asdescribed for RGC10 cells in U.S. Pat. No. 7,435,553, herebyincorporated by reference, Briefly, CHO K1 cells were stably transfectedwith vector pTE158 and pcDNA6/TR (Invitrogen). Transfected cells werescreened for doxycycline-inducible expression of hFcγR1, and one clonewas selected to give rise to the 3033 cell line. 3033 cells were adaptedto grow in suspension culture in serum-free Medium 3.

5055 Cells. 5055 cells are CHO K1 cells that have been adapted to growin suspension in serum-free bioreactor medium Medium 2.

4044 Cells. 4044 cells were derived from RGC16 cells described inInternational Patent Application Publication No. WO 2008/151219 A1 filed4 Jun. 2008, and US Patent Application Publication No. 2009/0124005A1filed 4 Jun. 2008, each hereby incorporated by reference, and whichcontains a laxed cassette at an enhanced expression and stability(EESYR) locus. The EESYR locus in 4044 has, from 5′ to 3′ on the codingstrand, a loxP site, an SV40 late promoter, a puromycin-resistance gene,a CMV promoter, an IRES, an eCFP gene, a lox2272 site, a CMV promoter, aDsRed gene, and a lox511 site. 4044 cells further contain a stablytransfected pcDNA6/TR vector.

Other Cells. The 7077 cell line was derived from 3033 cells without theuse of exogenous recombinant nucleic add. 6066, 8088, and 1010 celllines were derived from 4044 cells without the use of exogenousrecombinant nucleic acid.

Example 2: Production of Recombinant Antibodies in CHO Cells

Vectors. The vectors described herein have the features indicated, wherethe relative placement of the features is presented with respect to thecoding strand, listed 5′ to 3′.

pR4000: a human UbC promoter, a gene encoding the heavy chain of Ab 2,an SV40 late promoter, and a hygromycin resistance gene.

pR4001: a human UbC promoter, a gene encoding the light chain of Ab 2,an SV40 late promoter, and a puromycin resistance gene.

pR4002: a LoxP site, a human CMV promoter, a gene encoding the heavychain of Ab 2, an SV40 late promoter, and a Lox2272 site.

pR4003: a Lox2272 site, a hygromycin-resistance gene, an IRES, an EGFPgene, a human CMV promoter, a gene encoding the light chain of Ab 2, anda Lox511 site.

pR4004: an SV40 late promoter and the gene encoding Cre recombinase (seeWO 2008/151219A1, hereby incorporated by reference).

pR4005: a LoxP site, a human CMV promoter, a gene encoding the lightchain of Antibody 1 (Ab 1), a SV40 late promoter, and a Lox2272 site.

pR4006: a Lox2272 site, a hygromycin resistance gene, an IRES, an EGFPgene, a human CMV promoter, a gene encoding the heavy chain of Ab 1, anda Lox511 site.

pR4007: a LoxP site, a human CMV promoter, a gene encoding the lightchain of Ab 1, a SV40 late promoter, a gene encoding the N terminus ofthe hygromycin resistance protein, and a Lox2272 site.

pR4008: a Lox2272 site, a gene encoding the C terminus of hygromycinresistance protein, an IRES, an EGFP gene, a human CMV promoter, a geneencoding the heavy chain of Ab 1, and a Lox511 site.

pR4009: a LoxP site, a SV40 late promoter, a hygromycin resistance gene,an internal ribosome entry site (IRES), an EGFP gene, a human CMVpromoter, and a Lox511 site.

pR4010: a LoxP site, an SV40 late promoter, a hygromycin resistancegene, an IRES, an EGFP gene, a human CMV promoter, the wild type FXgene, and a Lox511 site.

pR4010: a LoxP site, an SV40 late promoter, a hygromycin resistancegene, an IRES, an EGFP gene, a human CMV promoter, and the mutated FXgene having mutations L289S and N90K.

Fucosylation proficiency in CHO cells was studied by cell surface LCAstaining and by analysis of recombinant antibodies produced from CHOcells. In one study, 7077 cells were used as the host cells for theexpression of Antibody 2 (Ab 2), a human IgG1 antibody against a human Bcell receptor, following a method described in U.S. Pat. No. 7,435,553,hereby incorporated by reference. Briefly, 1×10⁷ 7077 cells weretransfected with plasmid pR4000 (heavy chain, hygromycin resistance) andpR4001 (light chain, puromycin resistance) using Lipofectamine™(Invitrogen, Carlsbad, Calif.), The transfected cultures were selectedwith 400 micrograms/mL hygromycin and 10 micrograms/mL puromycin eachfor two weeks in F12 medium containing 10% fetal calf serum. Cells thatsurvived selection were pooled together and were adapted to grown insuspension in serum-free bioreactor medium Medium 2, Expression ofhFcγRI was induced by the addition of doxycycline to the culture mediumfor three days. The induced cultures were incubated with 1 milligram/mLrabbit IgG for 18 hours prior to staining with F(ab′)₂ fragment of agoat polyclonal FITC-conjugated anti-human Fc antibody (JacksonImmunoResearch, West Grove, Pa.). The cells were stained for 1 hour thenwashed twice with PBS prior to analysis by flow cytometry on a MoFlo™cell sorter (Fort Collins, Colo.). Cells with mean FITC fluorescenceintensity in the top 5% of the total cell population were sorted into apool and was named 7077-1 cells. 7077-1 cells were expanded for 10 daysin Medium 2. To produce recombinant Ab 2, 7077-1 cells were seeded at4×10⁵ cells/mL Medium 2 in a shaker flask at 37° C. Three days later,the conditioned medium was collected and the Ab 2 protein wherein waspurified by Protein A affinity chromatography.

4044 and 6066 CHO cells were used as host cells for the expression of Ab2 and Ab 1, a human IgG1 antibody against a human cytokine receptor.Briefly, to express Ab 2, 2×10⁶ 4044 and 2×10⁶ 6066 cells (each having aloxed cassette at an EESYR locus) were each transfected with pR4002(heavy chain in a loxed cassette), pR4003 (light chain and hygromycinresistance in a loxed cassette), and pR4004 (encodes Cre). To express Ab1, 2×10⁶ 4044 and 2×10⁶ 6066 cells were each transfected with pR4005(light chain in a loxed cassette), pR4006 (heavy chain and hygromycinresistance in a loxed cassette), and pR4004 (encodes Cre). Transfected4044 and 6066 cells were selected with 400 micrograms/mL hygromycin for10 days in F12 medium containing 10% FCS. Surviving cells were adaptedto grow in suspension in serum-free Medium 1 for seven days. Cells thathave undergone Cre-mediated cassette exchange at the EESYR locusexpressed EGFP but not DsRed or ECFP. Cells positive for EGFP butnegative for DsRed and ECFP were collected by cell sorting using aMoFlo™ sorter. The 4044-derived cells that were transfected with Ab 2and Ab 1 genes were designated 4044-2 and 4044-1 cells, respectively.The 6066-derived cells that were transfected with Ab 2 and Ab 1 geneswere designated 6066-2 and 6066-1 cells, respectively. 4044-2, 6066-2,4044-1, and 6066-1 cells were expanded by culturing in Medium 2 forseven days. To produce recombinant antibodies, the four cell lines wereseeded at 4×10⁵ cells/mL medium 2 in a shaker flask at 37° C. Three dayslater, the conditioned media were collected and the recombinantantibodies therein were purified by Protein A affinity chromatography.

8088 and 1010 CHO cells were also used as host cells for the expressionof Ab 1. Briefly, to express Ab 1, 2×10⁶ 8088 and 2×10⁶ 1010 cells wereeach transfected with pR4007 (light chain and first portion ofhygromycin resistance gene in a loxed cassette), pR4008 (heavy chain andsecond portion of hygromycin resistance gene in a loxed cassette), andpR4004 (encoding Cre). Transfected cells that survived selection with400 micrograms/mL hygromycin were adapted to grow in suspension inserum-free Medium 1. Cells that expressed EGFP but not DsRed or ECFPfrom the transfected 8088 and 1010 were isolated by cell sorting on aMoFlo™ and were designated 8088-1 and 1010-1. To produce Ab 1 protein,8088-1 and 1010-1 cells were seeded in shaker flasks at 4×10⁵ cells/mL.Three days later, the culture media were collected and the Ab 1 thereinwere purified by Protein A chromatography.

Example 3: Antibody Fucosylation Analysis

Purified human IgG1 antibody proteins were initially deglycosylaed withPNGase F under denatured condition (0.5% SDS, 2 mM TCEP, and blockedwith 1% NP-40) in 50 mM Tris pH 8.0 with protein/enzyme ratio of 1microgram/0.1 mU at 37° C. overnight. The released glycans were thenfluorescently derivatized with anthranilic acid at 80° C. for 1 hour.The samples were pre-cleaned to remove excess anthranilic acid reagentwith Waters Oasis™ HLB cartridges. The oligosaccharide mixture was thenanalyzed by reversed phase HPLC, using 0.5% TFA in ddH₂O as mobile phaseA, and 0.045% TFA in 90% acetontrile/10% ddH₂O as mobile phase B. Theglycans were resolved on a Thermo Hypercarb™ (Thermo Fisher, Waltham,Mass.) column (dimension of 100×2.1, particle size of 3 micrometers)through applying a gradient from 30 to 40% B over 40 minutes. Thesignals were detected using a fluorescence detector with an excitationwavelength of 230 nm, and emission wavelength of 425 nm. Furtheranalysis of the HPLC-separated glycan peaks through mass spectrometryrevealed that they were separated into two main groups; non-fucosylatedbi-antennary glycans and fucosylated bi-antennary glycans. Within eachgroup (fucosylated vs. non-fucosylated), the glycans were furtherseparated into digalactosyl (G2), monogalactosyl (G1) or agalactosyl(G0) forms. Integration of the peak area corresponding to differentglycan forms allow for the quantification on the populations of eachindividual glycans on the monoclonal antibody.

Example 4: Sequencing Major Transcripts of FX, GMD, GDP-FucoseTransporter, and FUT8 Genes

Proteins encoded by the FX, GMD, GDP-fucose transporter, and FUT8 genesare components the de novo fucosylation pathway. Sequences of the majortranscript of FX gene in CHO cell lines 5055, 4044-1, 7077-1, 6066-1,2121, 2020, 6069, 1010, and 8088 cells, and sequences of the majortranscript of the GMD gene in 4044-1, 6066-1, 1010, and 8088 cells weredetermined. Sequences of the major transcripts expressed from the FUT8and GDP-fucose transporter gene were also determined in 4044-1 and6066-1 cells.

Briefly, total RNA was isolated from 5×10⁶ CHO cells using Micro-FastTrack 2.0 Kit™ (Invitrogen, Carlsbad, Calif.). cDNAs for the fourfucosylation genes were synthesized from total RNA using Oligo-dT as theprimer and SuperScript III First-Strand Synthesis System™ (Invitrogen).GMD cDNA was PCR amplified using primers 5′-ctacaatctt ggtgcccaga gc-3′SEQ ID NO:7 and 5′-tccagttcag tttctgctgc g-3′ SEQ ID NO:8. FX cDNA wasPCR amplified using primers 5′-ttccctgaca agaccaccta tcc-3′ SEQ ID NO:9and 5′-tagttgtcgg tgaaccaggc ac-3′ SEQ ID NO:10. GDP-fucose transportercDNA was PCR amplified using primers 5′-gatgaggaca gcaggaacaa gc-3′ SEQID NO:11 and 5′-agcactcttc tcaccctctt tgg-3′ SEQ ID NO:12. FUT8 cDNA wasPCR amplified using primers 5′-agccaagggt aagtaaggag gacg-3′ SEQ IDNO:13 and 5′-ttgtagacag cctccatcct cg-3′ SEQ ID NO:14. The DNApolymerases used in the PCR reactions were a 20 to 1 mix of PlatinumTag™ (Invitrogen) and cloned Pfu (Stratagene, La Jolla, Calif.). PCRproducts were purified after gel electrophoresis and cloned into pCR2.1TOPO™ vector (Invitrogen) following the manufacturers instructions.Cloned DNA products were transformed into electro-competent DH10B cells.A minimum of three bacterial colonies from each transformation werepicked to inoculate three liquid cultures containing LB and 100micrograms/mL ampicillin. Plasmid DNAs in these cultures were purifiedusing QIAprep Spin Miniprep Kit™ (Qiagen). Sequences of the cloned PCRproducts were determined using the M13 primers located on the vector andthe respective 5′ and 3′ PCR primers. These sequences were compared toGenbank sequences for FX (accession number AF525365), GMD (accessionnumber AF525364), GDP-fucose transporter (accession number AB222037),and FUT8 (accession number BD359138) mRNA from C. griseus.

Mutations in consensus FX transcript sequences resulting in codonchanges from the Genbank reference sequence (AF525365) were identifiedin 7077-1, 6066-1, 2121, 2020, 6069, 1010, and 8088 cells (Table 1 and2). Sequences of the GMD transcripts from 4044-1, 6066-1, 1010, and 8088cells matched GMD sequences reported in GenBank (accession numberAF525364). Sequences of the GDP-fucose transporter and FUT8 transcriptsin 4044-1 and 6066-1 cells matched their respective sequences reportedin GenBank as well (accession numbers AB222037 and BD359138).

Example 5: Fucosylation in CHO Cell Ines with a Single L289S Mutation inthe FX Gene

Relative fucosylation proficiency in 3033, 4044, 6066, and 7077 cellswere first studied by staining the cells with the lectin Lens culinarisagglutinin (LCA). Briefly, 2×10⁶ 4044 and 6066 cells were each incubatedwith biotin-LCA (Vector Laboratories, Burlingame, Calif.) at 5micrograms/mL for one hour. After two washes with PBS, the cells wereincubated with phycoerythrin-conjugated streptavidin (JacksonImmunoResearch) for 30 minutes. The cells were then washed once with PBSand analyzed by flow cytometry. 3033 cells and 7077 cells were stainedwith FITC-LCA for one hour, washed twice, and analyzed by flow cytometry(FIG. 2). 3033, 4044, 6066, and 7077 cells were all stained by LCA. LCAstaining intensity on 6066 and 7077 cells were significantly weaker thanthe LCA staining intensity on 3033 and 4044 cells (FIG. 2), suggestingthat there was less protein fucosylation in 6066 and 7077 cells than in3033 and 4044 cells. To examine whether 6066 and 7077 cells could beused as host cells for the expression of hIgG1 antibodies with lowfucose content, 4044 and 6066 cells were stably transfected withexpression plasmids for Ab 2 and Ab 1, and 7077 cells were stablytransfected with expression plasmids for Ab 2 (see Example 2).Recombinant Ab 2 and Ab 1 were produced from the transfected cells inthree-day shaker flask cultures as well as in twelve-day fed-batchbioreactor cultures. Ab 2 and Ab 1 were purified from the conditionedmedia and the levels of their fucosylation were determined by HPLC(Table 1). As shown in Table 1, 7077-1, 6066-1, and 6066-2 producedrecombinant antibody with fucosylation level between 3.6% and 8% inshakers and bioreactors at 37° C.

TABLE 1 Host Production Fucosylation Fucosylation Cell Line Cell LineConsensus Reporter in shaker in bioreactor Designation Designation FXMutation Antibody (%) (%) 4044 4044-1 None Ab 1 89.3 85.8 7077 7077-1L289S Ab 2 4.0 4044 4044-2 None Ab 2 95 76.8 6066 6066-1 L289S Ab 1 7; 86.14; 6.86 6066 6066-2 L289S Ab 2 3.6

Example 6: Fucosylation in CHO Cell Lines with Two Amino Acid Changes inthe FX Gene

8088 and 1010 are two cell lines isolated from 6066 cells without theuse of exogenous, recombinant nucleic acid. 6069, 2020, and 2121 arethree cell lines isolated from 6066-1 cells without the use ofexogenous, recombinant nucleic acid. Sequences of the major transcriptfor FX gene were determined by RT-PCR (Table 2). These five cell lineswere found to have the same L289S mutation in 6066 and 7077 cells. TheFX transcripts in all five cell lines also carry mutations that changeone amino acid in addition to the L289S change. These mutations aresummarized in Table 2. 8088, 1010, 6069, 2020, and 2121 cells exhibitedreduced binding to LCA (Fla 4), suggesting reduced protein fucosylationin these cells.

To examine fucosylation proficiency in 8088 and 1010 cells, Ab 1 wasproduced from these two host cells by stable transfection. Thetransfected cultures were selected with 400 micrograms/mL hygromycin fortwo weeks, Cells that were resistant to hygromycin were adapted to growin Medium 1 in suspension cultures, Recombinant Ab 1 was produced inthree-day shaker flask cultures as well as in twelve-day fed-batchbioreactor cultures. Ab 1 was purified from the conditioned media andthe levels of Ab 1 fucosylation were determined by HPLC (Table 2). Asshown in Table 2, the transfected 8088 and 1010 cells producedrecombinant Ab 1 antibody with fucosylation level between 0.53% and0.96% in shakers and bioreactors at 34° C.

Fucosylation proficiency in 6069, 2020, 2121 cells was also examinedafter purification of Ab 1 protein produced in shaker flask cultures.Table 2 shows that these three cell lines produced Ab 1 withfucosylation levels ranging from 0.82% to 0.94%.

TABLE 2 Host Production Fucosylation Fucosylation Cell Line Cell LineReporter in Shaker in Bioreactor Designation Designation FX MutationProtein (%) (%) 8088 (8088) 8088-1 L289S, N90K Ab 1 0.96 0.71 10101010-1 L289S, G211R Ab 1 0.94 0.53 2121 L289S, P136L Ab 1 0.82 2020L289S, N79S Ab 1 0.94 6069 L289S, N79S Ab 1 0.87

Example 7: Fucosylation Proficiency in 6066-1 is Temperature-Dependent

The effect of culture temperatures on protein fucosylation in 6066-1cells was examined by LCA staining and by analysis of fucosylation of Ab1 protein produced from these cells. FIG. 5 shows the LCA stains of4044-1 and 6066-1 cells grown at both 37° C. and 34° C. 4044-1 cellsgrown at 34° C. and 37° C. were stained similarly by LCA. 6066-1 cellsgrown at 34° C. bound LCA at a level that was significantly higher than6066-1 cells grown at 37° C., Table 3 shows the level of fucosylation inAb 1 protein produced from 4044-1 and 6066-1 cells in shaker flaskcultures at 34° C. and 37° C. 4044-1 cells produced AB 1 with 96% and95°/h fucosylation when grown at 34° C. and 37° C. respectively. Incontrast, 6066-1 cells produced Ab 1 with about 70% and 7% fucosylationat 34° C. and 37° C. respectively. This result indicates that thefucosylation level in 6066-1 cells is temperature-dependent.

TABLE 3 Production Culture Fucosylation cell line Consensus Temp. in Ab1 Designation FX Mutation (° C.) (%) 4044-1 None 37 95 4044-1 None 34 966066-1 L289S 37 7 6066-1 L289S 34 70

Example 8: Fucosylation of CHO Cells Cultured in Media Supplemented withL-Fucose

In mammalian cells, GDP-fucose can be produced by the de novo synthesispathway and the salvage pathway (Becker and Lowe (2003) Fucose:biosynthesis and biological function in mammals, Glycobiology,13(7):41R-53R). In cells grown in culture medium lacking L-fucose,GDP-fucose is produced by GMD and FX proteins from GDP-mannose. Inmedium with L-fucose, GDP-fucose can be generated from L-fucose byL-fucose kinase and GDP-L-fucose pyrophosphorylase. GDP-fucose producedfrom either pathway is transported to the Golgi apparatus throughGDP-fucose transporter. In the Golgi, the fucosyltransferase proteinFUT8 converts glycoprotein into fucosylated proteins with GDP-fucose.Fucosylation proficiency of 6066-2, 8088, and 1010 cells grown inculture media with and without 5 mM L-fucose was examined. 6066-2 cellsexpressed the Ab 2 antibody and carried the L2893 mutation in the FXgene transcripts (Example 2 and Table 1). By HPLC analysis of purifiedAb 2, 6066-2 cells grown in shaker flasks produced AB 2 with 1.9%fucosylation in Medium 2 with no added L-fucose. In contrast, 6066-2cells grown in shaker flasks produced Ab 2 with 93.5% fucosylation inMedium 2 supplemented with 5 mM L-fucose. This result indicates that thesalvage pathway for GDP-fucose synthesis, the GDP-fucose transporter,and the FUT8 proteins were functional in 6066-2 cells.

The relative fucosylation proficiencies of 3033, 5055, 7077, 8088, and8088 cells grown in Medium 2 with and without 5 mM L-fucose wereexamined by staining with LCA (FIG. 6). 3033 and 5055 cells boundsimilar levels of LCA with and without L-fucose supplementation. 7077,8088, and 8088 cells bound significantly more LCA when grown in mediawith 5 mM L-fucose than in media lacking L-fucose. This result suggeststhat 7077, 8088, and 8088 cells had functional GDP-fucose transporterand functional FUT8 protein.

Example 9: Fucosylation in 8088 Transfected with FX Gene

To confirm that the reduced fucosylation level seen in 8088 cells wasdue to the mutant FX gene (with L289S and N90K mutations), wild type FXgene and the mutant FX gene were expressed in 8088 cells by stabletransfection and then fucosylation proficiency of the transfected cellswas examined by staining the cells with LCA. As a control, 8088 cellswere separately transfected with the vector pR4009 (laxed cassettehaving hygromycin resistance gene and EGFP gene). Vectors pR4010 andpR4011 contain the wild type FX gene and the L289S N90K FX gene placedin between the CMV promoter and the Lox511 site in pR4009, respectively.8088 cells transfected with pR4004 and either pR4009, pR4010, or pR4011were selected with 400 micrograms/mL hygromycin for 14 days. The cellsthat underwent Cre-mediated cassette exchange at EESYR expressed EGFPbut not EYFP. The cells that were EGFP-positive but EYFP-negative wereisolated by cell sorting. After expansion in tissue culture at 34° C.,the sorted cells were sequentially stained with biotin-LCA andPE-streptavidin. 8088 transfected with the vector pR4009 and pR4011exhibited the same level of LCA staining. In contrast, 8088 cellstransfected with pR4010 exhibited a level of LCA staining comparable to5055 cells (FIG. 7). In summary, wild type FX protein, but not the L289SN90K mutant FX protein, was able to restore the fuscosylation level in8088 cells as assayed by LCA staining. This result indicates that thelower fucosylation level in 8088 cells was due to the L289S N90Kmutation in FX protein in these cells.

Example 10: HPLC and Mass Spectrometry of Glycans: Abs 3.1 and 3.2

Cell line 8088, the CHO line having the FX gene modification that codesfor an FX protein substitution L289S and N90K, was separatelytransfected with plasmids encoding heavy (human IgG1) and light (humankappa) chains of two human antibodies with different variable regionsthat specifically bind the same growth factor receptor (Antibody 3.1 andAntibody 3.2). Cells expressing each antibody were grown in Medium 2 inthe presence and in the absence of 10 mM fucose for 3 days at 37° C.,and glycans from the antibodies under each set of conditions wereisolated and identified by mass spectroscopy.

8088 cells expressing A3.1 in the absence of fucose produced three majorglycan peaks on HPLC (FIG. 8), representing three differentnonfucosylated glycans on a mass spectrum that differed in terminalgalactosylation (FIG. 9) with about 1.47% fucosylation, 8088 cellsexpressing A3.1 in the presence of 10 mM fucose produced three majorglycan peaks on HPLC (FIG. 10), representing three different fucosylatedglycans and one nonfucosylated glycal on a mass spectrum (FIG. 11), withabout 95.22% fucosylation.

8088 cells expressing A3.2 in the absence of fucose produced three majorglycan peaks on HPLC (FIG. 12), representing three differentnonfucosylated glycans on a mass spectrum that differed in terminalgalactosylation (FIG. 13) with about 5.73% fucosylation. 8088 cellsexpressing A3.2 in the presence of 10 mM fucose produced three majorglycan peaks on HPLC (FIG. 14), representing three different fucosylatedglycans and a fourth minor amount of nonfucosylated glycan on a massspectrum (FIG. 15), with about 95.63% fucosylation.

Results of glycan analysis for fucose-fed and non-fucose fed 8088 cellsexpressing Antibody 3.1 or Antibody 3.2 are summarized in FIG. 16,grouped according to glycan type. Columns indicating percentage ofantibody under a particular condition sum to 100. For Ab 3.1, totalfucosylation in the absence of 10 mM fucose was 1.87%; for Ab 3.2, totalfucosylation in the absence of 10 mM fucose was 5.73% (sum thecorresponding columns in the last three rows of the table of FIG. 16).In the presence of 10 mM fucose, total fucosylation for Ab 3.1 was95.22%; in the presence of 10 mM fucose total fucosylation for Ab 3.2was 95.63% (sum the corresponding columns in final three rows of thetable of FIG. 16). These data establish that the low fucosylation celllines fucosylate no more than about 1.87% or 5.73% in the absence offucose, but that fucosylation can be recovered in the presence of fucoseup to at least about 95.22% or 95.63% fucosylation.

For glycan analysis, 100 microgram aliquots of each of the two antibody(Antibody 3.1 and Antibody 3.2) samples were resuspended in 45microliters of denaturation buffer containing 50 mM Tris (pH 8.0), 2.0mM tris(2-carboxyethyl)phosphine (TCEP), 0.5% SDS. The protein wasdenatured by heating at 80° C. for 7 min. The N-linked glycans on theantibody were released following incubation with 10 mU of PNGase F and1% NP40 at 37° C. overnight. The released glycans were fluorescentlylabeled by addition of 200 microliters of derivatization solution (30mg/mL anthranilic acid (AA) and 20 mg/mL sodium cyanoborohydride inmethanol containing 4% (w/v) sodium acetate and 2% (w/v) boric acid),and incubation at 80° C. for 1 hour. The AA derivatized glycans werefurther separated from excess reagents using a solid phase extractioncartridge (Oasis™ HLB cartridge, Waters Corp.) and eluted into 200microliters of 5% acetonitrile. For HPLC separation of the glycans, aThermo Hypercarb™ column (3 μm, 100×2.1 mm) was used at a flow rate of0.15 mL/min. Mobile phase A was 0.05% TFA in H₂O, and mobile phase B0.045% TFA in 90% acetonitrile and 10% H₂O. An aliquot of 10 microlitersof fluorescent derivatized oligosaccharides was mixed with 90microliters of 0.1% TFA in H₂O and injected onto the columnpre-equilibrated in 25% mobile phase B. Post sample injection, thegradient was increased to 30% B over 5 min, followed by another increaseto 43% B over 39 minutes to get the oligosaccharide separated. TheAA-labeled glycans were detected using a fluorescence detector withexcitation wavelength at 230 nm and emission wavelength at 450 nm. Themass spectrometry analysis of the AA-labeled glycans were conductedusing a Shimadzu Axima™ MALDI-TOF system. One hundred microliters of thederivatized glycans were dried in speed vacuum and resuspended in 10microliters 0.1% TFA. The concentrated glycans were further desaltedusing Nutip Hypercarb™, and eluted into 30 microliters of 0.1% TFA in65% acetonitrile, and speed vacuum dried. The lyophilized glycans wereredissolved in 2 microliters 10 mg/mL DHB (2,5-dihydroxybenzioc acid) in70% acetonitrile, and spotted onto the MALDI plate. The spectra werecollected under linear negative mode, with post extraction at 1500 mu,and laser power set between 60-90% of maximum power (6 mW) operated atwavelength of 337 nm.

1.-15. (canceled)
 16. A method of culturing cells comprising: a)providing mammalian cells which (i) comprise a salvage fucosylationpathway, (ii) comprise a de novo fucosylation synthesis pathwaycharacterized by a modifiedGDP-4-keto-6-deoxy-mannose-3,5-epimerase-4-reductase (FX) protein, and(iii) comprise and ectopically express a polynucleotide encoding anFc-containing glycoprotein, wherein the cells produce the Fc-containingglycoprotein in a fucosylated form when the cells are cultured in mediacomprising external fucose, and wherein the cells produce theFc-containing glycoprotein in a reduced fucosylated form when comparedto a mammalian cell comprising an unmodified Fx protein; and b)culturing the mammalian cells in a cell culture medium that: (i)comprises external fucose; or (ii) is devoid of external fucose.
 17. Themethod of claim 1, further comprising step c) isolating theFc-containing glycoprotein from the culture of step b)(i) or stepb)(ii).
 18. The method of claim 16, wherein the external fucose isL-fucose.
 19. The method of claim 16, wherein said culturing the cellsof step b)(i) comprises supplementing cell culture medium with 5 mMfucose or 10 mM fucose.
 20. The method of claim 16, wherein themammalian cells are selected from the group consisting of a COS, ChineseHamster Ovary (CHO), 293, BHK, HeLa and Vero cells.
 21. The method ofclaim 20, wherein the mammalian cells are CHO cells.
 22. The method ofclaim 16, wherein the Fc-containing glycoprotein is an antibody.
 23. Themethod of claim 16, wherein the modified FX protein comprises an aminoacid sequence that is at least 90% identical to SEQ ID NO:
 1. 24. Themethod of claim 23, wherein the modified FX protein comprises amino acidsubstitution L289S.
 25. The method of claim 24, wherein the modified FXprotein further comprises at least one additional amino acidsubstitution selected from the group consisting of N90K, N79S, P136L andG211R.
 26. The method of claim 24, wherein the modified FX proteincomprises amino acid substitutions L289S and N90K.
 27. The method ofclaim 16, further comprising detecting the amount of fucosylatedFc-containing glycoprotein from the culture of step b)(i) or stepb)(ii).
 28. The method of claim 27, wherein said detecting the amount offucosylated Fc-containing glycoprotein comprises: isolating theFc-containing glycoproteins from a sample of cells from the cell cultureof step b)(i) or step b)(ii); contacting the isolated glycoproteins witha label under conditions that permit the glycoproteins to bind thelabel; and detecting the amount of labeled glycoprotein in the sample byMass Spectrometry.
 29. The method of claim 27, wherein said detectingthe amount of fucosylated Fc-containing glycoprotein comprises:isolating the Fc-containing glycoproteins from a sample of cells fromthe cell culture of step b)(i) or step b)(ii); deglycosylating theglycoproteins to remove glycans from the glycoproteins; and detectingthe amount of fucosylated glycans in the sample by high performanceliquid chromatography (HPLC).
 30. The method of claim 27, wherein saiddetecting the amount of fucosylated Fc-containing glycoproteincomprises: staining a sample of cells from the cell culture of stepb)(i) or step b)(ii) with lectin; and detecting the amount of stainedcells in the sample to determine the amount of fucosylated glycoproteinin the sample.
 31. The method of claim 27, wherein at least 90% of saidFc-containing glycoproteins are fucosylated in step b)(i).
 32. Themethod of claim 31, wherein between 93% and 96% of said Fc-containingglycoproteins are fucosylated in step b)(i).
 33. The method of claim 27,wherein no more than 10% of the Fc-containing glycoproteins arefucosylated in step b)(ii).
 34. The method of claim 17, furthercomprising step d) determining the biological activity of the isolatedFc-containing glycoprotein.
 35. The method of claim 16, wherein saidculturing further comprises culturing the cells in the cell medium at37° C. for at least 3 days.